Capillary electrophoresis is a widely-used technique in high-throughput DNA sequencing. In practically all DNA sequencers, fluorescence labeling techniques are used for sequence detection. A number of extremely sensitive fluorescence detection techniques are available based on registering single photons, commonly referred to as the single photon detection (SPD) techniques. Such techniques are known in the prior art and are described in G. Papageorgas, H. Winter, H. Albrecht, et al., IEEE Trans. on Instrumentation and Measurement, 1999, Vol. 48, 6, pp. 1166-1177; Y. E. Tiberg and V. N. Paulauskas, Instruments and Experimental Techniques, 1981, Vol. 23, 5, pp. 1252-1255, A. M. Evtyushenkov, Y. F. Kiyachenko, G. I. Olefirenko and I. K. Yudlin, Instruments and Experimental Techniques, 1982, Vol. 24, 5, pp. 1265-1267, K. D. Shelevoi, Instruments and Experimental Techniques, 1985, Vol. 28, 3, pp. 614-616, the texts of which are fully incorporated herein by reference. Because of their complexity and cost, SPD techniques were mostly used for specialized scientific applications, as time resolved fluorescence spectroscopy or detection of single fluorescent molecules.
Recently, SPD techniques have been employed in single-lane DNA sequencing instruments, as documented in L. Alaverdian, S. Alaverdian, O. Bilenko, et al., I. Bogdanov, S. Domrachev, E. Filippova, D. Gavrilov, B. Gorbovitski, M. Gouzman, I. Gudkov, N. Lifshitz, S. Luryi, V. Ruskovoloshin, A. Stepukhovich, M. Tcherevishnik, G. Tyshko and V. Gorfinkel, Abstract Book of the conference “Advances in Genome Biology and Technology”, p. 47, Marco Island, Fla., USA, Feb. 3-6, 2001; and L. Alaverdian, S. Alaverdian, O. Bilenko, I. Bogdanov, E. Filippova, D. Gavrilov, B. Gorbovitski, M. Gouzman, G. Gudkov, S. Domratchev, S. Kosobokova, N. Lifshitz, S. Luryi, V. Ruskovoloshin, A. Stepoukhovitch, M. Tcherevishnick, G. Tyshko, V. Gorfinkel, Electrophoresis 2002, 23, pp. 2804-2817 (hereinafter “Alaverdian et al.”), the texts of which are fully incorporated herein by reference.
However, one of the most difficult challenges in the development of such systems is the elimination of the lane cross-talk caused by both optical and electronic cross-talk phenomena between channels of the single photon detector. The prior art systems have not addressed this issue.
There are two general types of lane cross-talk in single photon detection systems: optical and electronic. Electronic cross-talk may be caused by any multi-channel electronic module of a detection system, specifically as a result of, e.g., certain features of the electronic optics inside of a photo-multiplying tube. Optical cross-talk may be caused by, e.g., poor quality of a capillary array image on the receiving area of the photodetector, a contradiction between requirements of certain image magnification (from a lens) necessary for projection of the array as a whole onto the photodetector, and additional magnification of inner capillary volumes caused by capillary walls, a misalignment of the optical system after the capillary array placement, etc.
Cross-talk can be an especially significant problem in certain photodetection applications, such as, e.g., in DNA sequencing applications. Different lanes of a multi-lane DNA sequencer can have orders of magnitude variation in amplitude of fluorescent peaks. Accordingly, even very small lane cross-talk may cause ambiguity in data analysis.
Ultimately, some sources of channel cross-talk in a single photon detection system cannot be eliminated. Certain measures may be undertaken to reduce cross-talk, but these solutions are generally less efficient, more complex and expensive.
In prior art systems and methods, the general approach is to eliminate cross-talk, both optical and electrical, in the system. On the optical side, several strategies for removal of cross-talk have been employed. Examples include using an aperture mask to remove cross-talk at the input of the multi-capillary system, decreasing the distance of the collection system, and employing a smaller collection angle. However, while these measures may reduce or eliminate frond-end cross-talk, they also reduce the light collection efficiency. As a result, these cross-talk avoidance methods are limited in the types of equipment they may utilize, e.g., they may employ photodetectors which do not introduce cross-talk (even though these are typically the more sensitive and powerful photodetectors).
Accordingly, it is an object of the present invention to provide a novel system and method for calibration and elimination of channel cross-talk to enable an accurate separation of fluorescence signals emitted by individual capillaries.